Fig. 3: Lipid packing affects UBE2J2 conformation.
From: UBE2J2 sensitizes the ERAD ubiquitination cascade to changes in membrane lipid saturation
a Limited proteolysis assay of UBE2J2DL680 reconstituted into liposomes with indicated SFA content (P/L = 1:32,000), incubated with increasing trypsin concentrations (0, 1.9, 5.6, 16.6 or 50 ng/µl) at 25 °C for 30 min. Schematic (left) shows predicted tryptic sites (100% cleavage probability, PeptideCutter102; the red star indicates the position of the fluorescent label. Reaction products were analyzed by SDS-PAGE and fluorescence scanning. b Distribution of UBE2J2DL680 fluorescence along the gel lane for proteoliposomes incubated with 1.85 ng/µl trypsin from (a). c Fluorescence emission spectra of liposomes reconstituted with the FRET donor UBE2J2A488 in the absence or presence of the FRET acceptor UBE2J2A594 (P/L = 1:20,000 and 1:5000, respectively). Each condition overlays six curves (two reconstitutions, each measured in triplicate). Data were normalized to the emission at 516 nm. d Relative FRET efficiency calculated from spectra in (c) as the ratio of acceptor to donor emission intensity (I614/I516). e Cysteine crosslinking of UBE2J2DL680 in liposomes with the indicated SFA content (P/L = 1:32,000), incubated with the indicated concentrations of 1,4-bismaleimidobutane (BMB) or 1,2-bismaleimidoethane (BMOE). Samples were analyzed by SDS-PAGE and fluorescence scanning. f Ubiquitin loading of UBE2J2DL680, reconstituted in liposomes with 10% SFA content (P/L = 1:32,000), and the indicated proteins at different protein to lipid ratio (P/L). g Quantification of reactions in (f). For panels (a), (e), and (f), the experiments under these exact conditions were performed once; similar assays were repeated multiple times under related conditions and yielded consistent results. Detailed liposome compositions are given in Table 1. Source data are provided as a Source Data file.
